Homologous Recombination DNA Repair Research Articles

Selenocystine induces oxidative-mediated DNA damage via impairing homologous recombination repair of DNA double-strand breaks in human hepatoma cells

Selenocystine (SeC) has been identified as a novel compound with broad-spectrum anticancer activity. However, the effects of SeC on modifying DNA repair mechanism were less addressed. In this study, we demonstrated that SeC selectively induced cytotoxicity and genotoxicity against HepG2 hepatoma cell line. Comet assay revealed SeC-induced DNA damage in HepG2 cells, particularly in the form of DNA double strand breaks (DSBs), corroborated by the increase expression of the DSB marker, gamma-H2AX. We further demonstrated that SeC suppressed DNA homologous recombination repair, exacerbating DNA damage accumulation. Such effects on DNA damage and cell viability inhibition were alleviated by antioxidants, glutathione and Trolox, suggesting the involvement of reactive oxygen species (ROS). High levels of intracellular and mitochondrial ROS were detected in SeC-treated HepG2. In addition, SeC impaired the expression of antioxidant enzymes (superoxidase mutases and catalase), prompting the imbalance between antioxidant protection and excessive ROS formation and eliciting DSBs and cellular death. Decreased procaspase-3, 7, and 9 and Bcl-2 proteins and an increased Bax/Bcl-2 ratio, were observed after SeC treatment, but could be reversed by Torlox, confirming the action of SeC on ROS-induced apoptosis. In vivo, the xenograft tumor model of HepG2 cells validated the inhibition of SeC on tumor growth, and the induction of DSBs and apoptosis.In summary, SeC has the capability to induce ROS-dependent DNA damage and impeded DBS repair in HepG2 cells. Thus, SeC holds great promise as a therapeutic or adjuvant agent targeting DNA repair for cancer treatment.

Research progress on the function and mechanism of CXorf67 in PFA ependymoma

<p indent="0mm">CXorf6 (chromosome X open reading frame 67) protein contains 503 amino acids and is predicted <italic>in silico</italic> to be mostly disordered. <italic>CXorf6</italic>7 is barely expressed in most cell lines and human tissues. Therefore, for rather a long time, our understandings regarding functions of this protein are quite limited. In 2018, Pajtler et al. discovered a high expression of <italic>CXorf67</italic> specifically in posterior fossa A (PFA) ependymomas. After that, CXorf67 attracts increasing attention and efforts to explore its potential roles in PFA ependymomas. Based on DNA methylation profiling, ependymomas have been classified into 9 molecular subgroups. Of these 9 subgroups, PFA is one of the most malignant and aggressive ones with the highest incidence. PFA ependymomas mostly happen in infants and young children with a median age of <sc>~3 years old,</sc> and are generally refractory to chemotherapies. Current standard therapy for intracranial ependymomas is surgical resection combined with radiotherapy. However, the lateral localization of PFA ependymomas renders them hard to be removed completely, and together with a lack of chemotherapy options, they generally have a poor prognosis. Research from different labs has so far revealed two independent functions of CXorf67 in PFA ependymomas. One function of CXorf67 is related to the inhibition of PRC2 (Polycomb repressive complex 2) enzymatic activity and thus a global H3K27me3 reduction in PFA ependymomas. Results from several studies consistently suggested that CXorf67 functions as an inhibitor of PRC2 through an H3K27M-like mechanism. These findings implicate CXorf67 as a promising therapeutic target for PFA ependymomas, and efforts to develop drugs based on this PRC2-related function of CXorf67 remain on the way. On the other hand, a recent study revealed the other function of CXorf67 that is involved in DNA damage repair—CXorf67 inhibits DNA homologous recombination repair through blocking PALB2-BRCA2 interaction. This new function of CXorf67 renders it as a biomarker for PARP inhibitor treatment and provides a new treatment option for PFA patients with immediate clinical applicability. In this review, we summarize current findings about the functions and mechanisms of CXorf67, as well as the possible regulatory mechanisms of <italic>CXorf67</italic> expression in PFA ependymomas. We also discussed the clinical implications of the two functions of CXorf67 for PFA ependymomas. In addition to PFA ependymomas, <italic>CXorf67</italic> expression is also observed in diffuse intrinsic pontine gliomas (DIPG) and germ cell tumors. We believe that it will be of great significance to investigate whether <italic>CXorf67</italic> expression could also sensitize these two tumors to PARP inhibitors. Moreover, <italic>CXorf67</italic> is specifically unregulated in PFA, but not in any other ependymoma subgroup; and reasons for this remain currently unclear. Understanding of how <italic>CXorf67 </italic>expression is turned on in PFA ependymomas might enable a controllable expression of <italic>CXorf67</italic> in other types of tumors, thereby opening up new opportunities not only for PFA patients, but possibly also for other cancer patients.

Opinion: PARP inhibitors in cancer-what do we still need to know?

PARP inhibitors (PARPi) have been demonstrated to exhibit profound anti-tumour activity in individuals whose cancers have a defect in the homologous recombination DNA repair pathway. Here, we describe the current consensus as to how PARPi work and how drug resistance to these agents emerges. We discuss the need to refine the current repertoire of clinical-grade companion biomarkers to be used with PARPi, so that patient stratification can be improved, the early emergence of drug resistance can be detected and dose-limiting toxicity can be predicted. We also highlight current thoughts about how PARPi resistance might be treated.

Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

Abstract 1280: Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients

Abstract Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) are becoming the standard of care for ovarian cancer. The greatest benefit has been observed in patients who have mutations in BRCA1/2 or related genes in the homologous recombination (HR) DNA repair pathway. Nonetheless, responses to PARPi were not durable, indicating that strategies to prevent recurrence and overcome resistance, such as combinations with immune checkpoint inhibitors (ICIs), are of high priority. However, most studies on PARPi have focused on the tumor itself and synthetic lethality. The immunological effect, particularly the effect on adaptive immune response, has been overlooked. PARP is expressed in almost all cells, and it regulates the transcription of various genes through poly(ADP-ribosyl)ation. Therefore, we hypothesized that PARPi affect the composition and functions of T cells in the maintenance therapy setting, and consequently reconstitute the anti-tumor immune response in ovarian cancer. Here, we collected serial peripheral blood mononuclear cells (PBMCs) from PARPi-treated patients and analyzed their dynamic immunologic changes using muticolor flow cytometry. The PBMCs were collected during PARPi maintenance therapy, and the time points included pre-treatment as well as 1 month and 3 months after the initiation of treatment. Olaparib or niraparib was used as maintenance therapy. First, the percentages of CD4, CD8, and FoxP3+regulatory T cells (Treg cells) were compared at each time point and by PARPi type. Interestingly, the proportion of Treg cells increased at 1 and 3 months after initiation, especially in the niraparib group. However, there were no significant changes in the proportion of CD4 and CD8 T cells. Second, we analyzed immune checkpoints and properties in each T cell population. We found that the PD-1 expression on Treg cells decreased after 1 and 3 months. Although the percentage of Treg cells increased, proliferative Ki-67+Treg cells decreased after 1 and 3 months, especially in the niraparib group. Third, intracellular staining assay was performed to measure functional changes of T cells during PARPi treatment. IFN-γ and/or TNF-α producing CD8 and CD4 T cells decreased after 1 month compared to pre-treatment. Finally, proliferative Ki-67+CD8 T cells and PD-1+CD8 T cells decreased after 72 hours of in vitro olaparib treatment compared to the control group. In conclusion, long-term PAPRi treatment may affect Treg cells and CD8 T cells. However, strategies to overcome negative effects of PARPi and their detailed mechanisms need to be further investigated. Additional data in this regard will be presented at the meeting. Citation Format: Junsik Park, Miran Lee, Yup Kim, Yoo-Na Kim, Yong-Jae Lee, Jung-Yun Lee. Immunologic effect of PARP inhibitors as maintenance therapy in ovarian cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1280.

Abstract 5103: Characterization of the genomic landscape and actionable mutations in Taiwanese prostate cancer patients

Abstract Background: Prostate Cancer (PCa) is the second most common male cancer worldwide and the most common cancer in males over 65 years old. PCa is the sixth most common cancer in Taiwan, and its incidence rate has increased gradually over the past 30 years. PAPR inhibitors are the only type of targeted drug approved by the FDA to treat advanced castration-resistant prostate cancer (CRPC) that has grown after the hormone therapy drugs. This study aimed to characterize the genomic profile of Taiwanese PCa and explore whether there are potential treatment guidances derived. Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissues from 99 patients diagnosed with PCa and collected at Chi-Mei Medical Center (CMMC) were subject to next-generation sequencing using ACTOnco® CGP panel that cover coding regions of 440 cancer-related genes. Genomic alterations (GAs) and signatures, including single nucleotide variations (SNVs), short insertions and deletions (InDels), copy-number variations (CNVs), fusion genes, tumor mutations burden (TMB), and microsatellite instability (MSI) status were retrieved. The gene mutation prevalence was compared with the Western population using the TCGA prostate cancer dataset. Results: A total of 1558 variants was identified, including 215 SNVs, 22 InDels, 1178 CN gain and 135 homozygous deletions. Similar to the Western population, the most frequently mutated genes were SPOP (21.2%), KMT2C (10.1%), MUC16 (8.1%), TP53 (6.1%), ARID2 (5.1%), IDH1 (5.1%), PTGS2 (5.1%). Further analysis showed that 12 patients harbored deleterious/likely deleterious mutations in genes implicated in homologous recombination (HRR) DNA repair pathway, including three BRCA2, two CDK12, BRAD1 and ARID1A, respectively, and one ATR, ATM and PALB2, respectively. All BRCA2 mutations were predicted to be germline origin and only one-third of 12 HRR mutants had a biallelic gene loss. Median TMB calculated from NGS data is 0.6 muts/Mb (range 0 to 4.5) and all samples are microsatellite-stable (MSS). Therapeutically actionable gene and pathway analysis revealed that up to 24.2% of patients harbored at least one aberration against FDA-approved targeted drugs, such as MET copy number (CN) gain (11.1%), mTOR pathway aberration (6.1%), HRR gene biallelic loss (4%), BRAF activating mutation (4%), IDH1 activating mutations (4%), EGFR CN gain (3.0%), FGFR1 CN gain (2.0%), MET fusion (1.0%) and BRAF fusion (1%). Conclusion: This CGP study unraveled the genomic profile of Taiwanese prostate cancers, and the results showed that GAs identified could serve as a potential biomarker to guide personalized, targeted therapies in one-fourth of patients. Citation Format: Kien Thiam Tan, Chia-Ling Wu, Yen-Jung Lu, Ka-Po Tse, Chien-Feng L Li. Characterization of the genomic landscape and actionable mutations in Taiwanese prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5103.

Abstract 5692: Tissue specificity of chromosome aneuploidy correlates with BRCA-associated cancer risk

Abstract Pathogenic germline variants of BRCA1 and BRCA2 disproportionally elevated the risk of specific cancer types (ex. breast [BRCA] and ovarian [OV] cancer) compared to tumors arising from other tissues. The reason underlying the strong tissue-specificity for BRCA-associated cancer risk remains largely unknown. Under the two-hit hypothesis, BRCA-mediated oncogenesis is thought to originate from a cell where the germline BRCA1/2 variant underwent loss of heterozygosity (LOH) of the wildtype BRCA allele, resulting in homologous recombination DNA repair deficiencies. Notably, tumor aneuploidy and loss of chromosome arms also show strong tissue specificity across cancer types. The apparent tissue specificities of BRCA-associated cancer risk and chromosome aneuploidy, together with deletion-induced LOH underlying the two-hit hypothesis, pose an intriguing possibility that they may be linked. Using genomic data from the TCGA PanCanAtlas and ICGC PCAWG projects, we calculated the frequencies of deletions of the chromosomal arms where the BRCA1/2 genes are located (17q for BRCA1; 13q for BRCA2) across different cancer types in tumors with no whole-genome doublings (WGD). Among TCGA cases, OV and BRCA showed significantly higher frequencies of 17q deletion (32.75% and 8.05%, respectively) than other cancer types (ranked 2/30 and 5/30, permutation p-value = 0.030 and 0.024), and relatively higher frequencies of 13q deletion (32.75% and 21.92%, respectively) compared to other cancers (ranked 5/32 and 8/32, p-value = 0.101 and 0.078). As expected, cancer types showing high fractions of BRCA1/2 deletions overlap with those showing 17q/13q deletions and often include BRCA and OV. Subsequently, we determined how often BRCA1/2 deletion co-occurred and thus may be caused by the corresponding 17q/13q deletions, showing that BRCA1 deletion carriers who also had chr17q deletion accounted for 40.76% in OV and 25.93% in BRCA patients, compared to 33.33% in all other cancer types while BRCA2 deletion carriers who also had chr13q deletion accounted for OV (53.97%) and BRCA (58.60%) patients, compared to 49.83% in all other cancer types. In addition, we validated our findings in non-overlapping cases of the ICGC PCAWG projects. Consistent with TCGA results, OV and BRCA showed higher frequencies of 17q and 13q deletion compared to other cancer types. To conclude, we identified a correlation between cross-cancer difference in arm-level and focal chromosome aneuploidy affecting BRCA1/2 and BRCA-associated cancer risk, and how chromosome aneuploid may give rise to tissue-specific risks of cancer remain to be investigated. Citation Format: Xinfeng Wang, Tomi Jun, Nan Sun, Jie He, Kuan-lin Huang. Tissue specificity of chromosome aneuploidy correlates with BRCA-associated cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5692.

Abstract 2590: Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma

Abstract Objective: Outcomes for patients with metastatic or relapsed osteosarcoma (OS) remain poor. Many OS samples have demonstrated a high proportion of mutational signature 3 (ms3), suggestive of homologous recombination DNA repair deficiency (HRD) or a BRCAness molecular phenotype. The HRD score, which quantifies chromosomal structural rearrangements by combining loss of heterozygosity (LOH), large state transitions (LST), and telomere allelic imbalances (TAI), has been used to assess HRD status in patients with breast and ovarian cancer, with high scores found to be predictive of PARP inhibitor sensitivity. While high HRD scores have been described in OS, little is known about how HRD scores vary by stage, site, pre vs post-therapy, presence of homologous recombination pathway or TP53 alterations, and their correlation to ms3 contribution. Methods: To address this issue, we evaluated 48 OS samples with tumor purity ≥ 20% from 46 patients who had 80x whole genome sequencing (WGS) performed through our pediatric expanded genomics platform. HRD scores were then estimated using scarHRD, a computational tool, which quantifies LOH, LST, and TAI events from WGS data and has shown a good correlation to SNP-array based methods. Results: Of the 48 samples analyzed, 25 samples were from primary sites and 23 from metastatic sites of disease. The median HRD score was 45 with a minimum score of 6 and a maximum score of 79. The 25th, 75th, and 90th percentiles of values were 35.7, 57, and 66.3 respectively. There was a significant difference in HRD score in patients with localized disease (n=22) vs metastatic disease (n=26) at time of diagnosis with median HRD (mHRD) scores of 41 and 51.5 (p = 0.027). The presence of a TP53 structural variant or single nucleotide variant (mHRD=47) was associated with higher HRD scores than TP53 wild-type samples (mHRD=29.5, p=0.04). There was no statistically significant difference in HRD scores in pre vs post-treated samples or primary site vs metastatic site samples. Samples with homologous recombination pathway alterations (n=10) were not associated with increased HRD score. There was a positive correlation (Spearman’s rho=0.425, p=0.001) between the number of mutations attributed to ms3 and HRD score. Conclusions: The range and distribution of HRD scores in our OS cohort closely resemble BRCA1/2 deficient breast cancer. Patients with metastatic disease at diagnosis were found to have higher HRD scores, representing a higher degree of genomic instability and increasing potential to accumulate resistance mutations. Higher HRD scores in TP53 mutated samples may be due to chromosomal instability caused by mechanisms other than HRD. The BRCAness phenotype in OS is now being targeted in a phase II clinical trial, combining the PARP inhibitor olaparib with the ATR inhibitor ceralasertib (NCT0441706), which includes integrated correlative biology to prospectively assess biomarkers of response such as HRD score. Citation Format: Michael David Kinnaman, Julia Pena, Max Levine, Nancy Bouvier, Elli Papaemmanuil, Suzanne Forrest, Katherine Janeway, Paul Meyers, Julia Glade Bender. Analysis of homologous recombination deficiency (HRD) scores in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2590.

Abstract 3249: Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer

Abstract PARP inhibitors (PARPi) offer major clinical benefit for patients with ovarian or other cancers, especially those with deficiencies in the homologous recombination DNA repair pathway. However, most patients with advanced cancer eventually acquire resistance to PARPi, and the mechanisms by which tumors escape PARPi therapy are still not fully understood. Here, we report that treatment with the PARPi olaparib induces the formation of polyploid giant cancer cells (PGCCs) in ovarian and breast cancer cell lines (Hey, SKOV3, MCF-7), three human high-grade serous cancer (HGSC)-derived organoids, and HGSC patient-derived xenografts. Live-cell fluorescence time-lapse tracking of ovarian cancer cells labeled with FUCCI (fluorescent ubiquitination cell-cycle indicator) or histone H2B-mCherry/α-tubulin-EGFP revealed that PGCCs primarily developed from the endoreplication of diploid cancer cells after exposure to sublethal concentrations of olaparib. PGCCs exhibited markers of senescent cells; however, they were able to escape senescence to generate mitotically competent daughter cells via budding, multipolar mitosis, and acytokinetic mitosis following olaparib withdrawal. PGCCs and derived daughter cells conferred resistance to olaparib-induced cytotoxicity, which could be blocked by the anti-progesterone drug mifepristone. Whole transcriptome analysis by RNA sequencing of ovarian cancer cell lines and ovarian cancer-derived organoids showed activation of a senescence-associated secretory phenotype with upregulated cytokines and chemokines and downregulation of MYC signaling in PGCCs. Mifepristone/olaparib combination therapy significantly mitigated tumor growth in patient-derived xenograft models with acquired resistance to olaparib. Thus, targeting of PGCCs may represent a promising approach to overcome PARPi resistance in recurrent ovarian cancer. Citation Format: Xudong Zhang, Jun Yao, Xiaoran Li, Na Niu, Yan Liu, Anil K. Sood, Jinsong Liu. Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3249.

Deep Sequencing of Early T Stage Colorectal Cancers Reveals Disruption of Homologous Recombination Repair in Microsatellite Stable Tumours with High Mutational Burdens.

Simple SummaryStage IIIA are rare early T stage colorectal cancers that are not widely reported in genome sequencing studies. We aimed to investigate genomics features in common cancer genes associated with lymph node metastasis from these tumours. We discovered that microsatellite stable tumours with high mutational burdens showed different genomic variant frequencies compared to tumours with low mutation burdens. The high burden microsatellite stable tumours showed co-occurrence of mutations in homologous recombination DNA repair genes that where not observed in the microsatellite stable cancers with low mutational burdens. This finding highlights a potential therapeutic approach for high mutation burden microsatellite stable colorectal cancers given the successful clinical implementation of drugs blocking homologous recombination DNA repair in other cancers.Early T stage colorectal cancers (CRC) that invade lymph nodes (Stage IIIA) are rare and greatly under-represented in large-scale genomic mapping projects. We retrieved 10 Stage IIIA CRC cases, matched these to 16 Stage 1 CRC cases (T1 depth without lymph node metastasis) and carried out deep sequencing of 409 genes using the IonTorrent system. Tumour mutational burdens (TMB) ranged from 2.4 to 77.2/Mb sequenced. No stage-related mutational differences were observed, consistent with reanalysis of The Cancer Genome Atlas (TCGA) Stage I and IIIA datasets. We next examined mutational burdens and observed that the top five cancers were microsatellite stable (MSS) genotypes (mean TMB 49.3/Mb), while the other 16 MSS cancers had a mean TMB of 5.9/Mb. To facilitate comparison with TCGA hypermutator CRC, we included four microsatellite instability-high (MSI-H) samples with the high mutation burden MSS cases to form a TMB-High group. Comparison of TMB-High with TMB-Low groups revealed differences in mutational frequency of ATM, ALK, NSD1, UBR5, BCL9, CARD11, KDM5C, MN1, PTPRT and PIK3CA, with ATM and UBR5 validated in reanalysis of TCGA hypermutator Stages I and IIIA samples. Variants in ATM were restricted to the TMB-High group, and in four of five MSS specimens, we observed the co-occurrence of mutations in homologous recombination repair (HRR) genes in either two of ATM, CDK12, PTEN or ATR, with at least one of these being a likely pathogenic truncating mutation. No MSI-H specimens carried nonsense mutations in HRR genes. These findings add to our knowledge of early T stage CRC and highlight a potential therapeutic vulnerability in the HRR pathway of TMB-H MSS CRC.

Germline genetics of prostate cancer.

An important fraction (>/~10%) of men with high-risk, localized prostate cancer and metastatic prostate cancer carry germline (heritable) pathogenic and likely pathogenic variants (also known as mutations) in DNA repair genes. These can represent known or suspected autosomal dominant cancer predisposition syndromes. Growing evidence suggests that pathogenic variants in key genes involved in homologous recombination and mismatch DNA repair are important in prostate cancer initiation and/or the development of metastases. Here we provide a comprehensive review regarding individual genes and available literature regarding risks for developing prostate cancer, and discuss current national guidelines for germline genetic testing in the prostate cancer population and treatment implications. The association with prostate cancer risk and treatment implications is best understood for those with germline mutations of BRCA2, with emerging data supporting associations with ATM, CHEK2, BRCA1, HOXB13, MSH2, MSH6, PALB2, TP53 and NBN. Treatment implications in the metastatic castration resistant prostate cancer setting include rucaparib and olaparib, and pembrolizumab with potential clinical trial opportunities in earlier disease settings. The data summarized in this review has led to the expansion of national guidelines for germline genetic testing in prostate cancer. We review these guidelines, and discuss the importance of cascade genetic testing of relatives, diverse populations with attention to inclusion, as well as prostate cancer screening updates and clinical trial opportunities for men who carry genetic risk factors for prostate cancer.

DOLAF: An international multicenter phase II trial of durvalumab (MEDI4736) plus olaparib plus fulvestrant in patients with metastatic or locally advanced ER-positive, HER2-negative breast cancer selected using criteria that predict sensitivity to olaparib.

TPS1116 Background: PARP inhibitors have documented clinical activity in patients with HER2 negative breast cancer (BC) and a germline pathogenic variant (PV) in BRCA1 or BRCA2. Defects in other genes involved in homologous recombination DNA repair (HRR) or mismatch repair pathway (microsatellite instability MSI) have been associated with preclinical cellular sensitivity to PARP inhibitors. Several preclinical and clinical studies have suggested synergy between immune checkpoint blockade and PARP inhibitors. Indeed, tumors with deficiency in HRR have higher mutagenic potential and produce a larger number of neoantigens. Around 60% of BC with a germline PV in BRCA1/ 2 are ER+/HER2- tumors, and the ER-pathway remains a key target of their therapy. The combination of PARP inhibitors with endocrine therapy has shown to be superior to monotherapy. Methods: DOLAF is an open-label, international, multicentric, phase II trial assessing the combination of olaparib, fulvestrant, and durvalumab in ER+/HER2- metastatic or locally advanced BC with somatic or germline PV in BRCA1, BRCA2 or other genes implicated in the HRR pathway (ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FAND2, FANCL, MRE11A, NBN, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and RAD54L) or in MSI status or other actionable genes ( AKT1, ESR1, FGFR1, FGFR2, FGFR3, and PIK3CA) all based on central tumor NGS. Further an amendment in May 2021, patients with only alterations in these other actionable genes can no longer be included. Patients must have received 1 prior line of endocrine therapy for their metastatic BC, including CDK4/6 inhibitor and maximum of 1 line of chemotherapy in the metastatic setting. Patients receive olaparib (twice daily at 300 mg), fulvestrant (2 intramuscular injections of 250 mg every 28 days) and durvalumab (1500 mg intravenous every 4 weeks). The primary objective is to evaluate the progression-free survival rate at 24 weeks. Secondary endpoints include safety, overall survival, objective response rate, in the overall population and in the germline BRCA mutated population. With an optimum two-stage Simon design, α = 2.5%, β = 5%, p0 (probability of inefficiency maximum) = 50%, p1 (probability of minimum efficiency) = 65%, it is necessary to include 158 patients. The strategy could be considered sufficiently effective if there are at least 87 patients without progression at 24 weeks. Given the lack of safety data from this association, a safety run-in phase including 6 patients has been completed without DLT. As of December 31, 2021, 266 patients have been screened of whom 102 have been treated. The first interim analysis occured in November 2021 after the inclusion of 64 evaluable patients. IDMC suggested that the trial continue as planned. Clinical trial information: NCT04053322.

SIK2 inhibition enhances PARP inhibitor activity synergistically in ovarian and triple-negative breast cancers.

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7–associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.

Results of the safety run-in of gedatolisib plus talazoparib in advanced triple negative or BRCA 1/2 positive HER2 negative breast cancer.

e13075 Background: Metastatic triple negative breast cancer (TNBC) is a disease subtype with a poor prognosis, though advancements have been made in the use of targeted therapies, such as poly (ADP-ribose) polymerase inhibitors (PARPi) for those with germline BRCA 1/2 mutations. Up to 2/3 of TNBC tumors have acquired defects in homologous recombination (HR) DNA repair, yet PARPi monotherapy has been ineffective in extending survival in these patients. Phosphoinositide-3-kinase (PI3K)/mTOR pathway alterations are also common, and preclinical data supports that PI3K/mTOR inhibition may disrupt normal function of the HR complex and increase dependency on PARP enzymes for HR DNA repair. Thus, combining a PI3K/mTORi with a PARPi may result in a synergistic anti-neoplastic effect. Methods: The safety run-in portion of this study evaluated the safety and preliminary efficacy of the combination of weekly IV gedatolisib (PI3K/mTORi) and continuous daily talazoparib. Germline BRCA mutations were not required. The safety run-in was designed to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). A 3+3 design was utilized for dose escalation, with starting dose at level 1. Dose levels are as shown in the Table. Eligibility criteria included patients ≥ age 18 who had received 1-3 prior lines of therapy for advanced TNBC or advanced HER2-negative BC with a germline BRCA 1/2 mutation. Those with untreated CNS metastasis were excluded as were those type I diabetes or uncontrolled type II diabetes, due to the risk of hyperglycemia with gedatolisib. Results: A total of 14 female patients were enrolled on to the safety run-in phase of the trial. Median age was 53 (range 30-67). Most patients (79%) were Caucasian, 14% were African American, and 7% unknown. The most common adverse events (AEs) of any grade were fatigue, anemia, nausea, and oral mucositis. Grade 1 hyperglycemia was noted in about 1/3 of the cohort, with 1 grade 3 event. There were 3 patients who experienced grade 4 AEs, thrombocytopenia (2) and lymphopenia (1). There was 1 DLT of grade 3 neutropenia which occurred at dose level 1. The MTD of gedatolisib was 180 mg and MTD of talazoparib was 1 mg. In this preliminary cohort, 3 patients achieved PR and 5 patients achieved SD. Median duration of PR was 13.34 months and median duration of SD was 4.11 months. Of the 3 patients with a BRCA 1 or 2 mutation, 2 had a best response of PR and the other SD. Conclusions: The safety run-in indicated that this combination is safe and well tolerated with mostly grade 1-2 AEs. This combination therapy has moved into the phase II trial, with 2 cohorts. Cohort A includes BRCA-wildtype patients with TNBC and cohort 2 includes those with a BRCA mutation and HER2-negative disease. Clinical trial information: NCT03911973. [Table: see text]

Correlative results from NCI protocol 10250: A phase II study of temozolomide and olaparib for the treatment of advanced uterine leiomyosarcoma.

11509 Background: uLMS is an aggressive sarcoma subtype of smooth muscle origin. Chemotherapy provides limited benefit for advanced disease. 18-25% of uLMS harbor deleterious alterations in homologous recombination (HR) DNA repair genes. uLMS exhibits high levels of replicative stress. These findings prompted a phase 2 study of O+T in pretreated uLMS where O+T demonstrated activity: ORR 27%, mPFS 6.9 mos (Ingham M. et. al. ASCO 2021: #11506) Methods: NCI protocol #10250 is a single-arm, multicenter, phase 2 trial evaluating O+T in advanced uLMS pts with progression on ≥1 prior line. Pre-treatment (Pre) and on-treatment (On) biopsies were collected from 22 pts. In prespecified analysis, we evaluated for a relationship between clinical outcomes and HR gene alterations by whole exome sequencing (WES), SLFN11/MGMT expression by RNAseq, and RAD51 foci formation (functional assay). HRD scores were calculated from WES using scarHRD. Gene expression was evaluated using a Spearman rank-order correlation analysis to identify genes associated with PFS (p &lt; 0.01) and overexpressed in sensitive (S: PFS &gt; 240d) or resistant (R: PFS &lt; 240d) pts. Gene set enrichment analysis (GSEA) was performed (q = FDR-adjusted p value). Pts with available results: WES/RNAseq (16), Pre HRD score (13), Pre RAD51 foci (12). Results: 31% (5/16) pts had a mutation (Mut) or homozygous deletion (Hd) in the HR panel: ATRX Mut (2), ATR Mut, PALB2 Hd, RAD51B Hd. Pts with PALB2 and RAD51B Hd had longest PFS on study. Recurrent alterations also occurred in TP53 (56%) and RB1 (19%). Median HRD score in Pre samples was 51 (range 36-66) and 10/13 had HRD scores ≥ 42. Pre and On SLFN11 and MGMT RNA expression were not correlated with ORR/PFS. 6/13 Pre samples were HR-deficient by the RAD51 foci assay. Of pts with PFS ≥ 200d, 4/6 were HR-deficient. In Pre samples, 81 genes were overexpressed in S pts and 73 in R pts. In On samples, 146 genes were overexpressed in S pts and 127 in R pts. In On samples, GSEA identified the epithelial-mesenchymal transition enriched in S pts (q = 3.38e-7) and cell cycle pathways (E2F targets, G2M checkpoint) in R pts (q = 7.43e-4). Only 2 genes, CXCL10 and PCDH15, were differentially expressed between paired Pre and On samples (both increased in On). Gene expression signatures for replicative stress showed borderline association with worse PFS. Conclusions: Most uLMS tumors exhibit HR defects as measured by HRD scores. A subset of pts with greater benefit from O+T were identified by WES for HR genes and the RAD51 assay. There was no correlation between SLFN11 and MGMT expression and outcomes. GSEA identified pathways differentially expressed in S and R pts in On samples. O+T induced CXCL10 which has been associated with T-cell trafficking to tumors. A randomized phase 3 trial of O+T versus investigator’s choice is planned. These results provide insight into which pts may benefit most from this novel drug combination. Clinical trial information: NCT03880019.

Synergistic activity of PARP inhibitors (PARPi) in combination with standard chemotherapy (CTx) in leiomyosarcoma.

11560 Background: Leiomyosarcomas (LMS) are genetically heterogeneous tumors that arise from smooth muscle. Currently, the mainstay of systemic treatment for patients with advanced/metastatic disease is doxorubicin (Dox) based CTx. Several genomic analyses of LMS reveal defects in homologous recombination (HR) DNA repair pathway in about half of patients, consistent with a druggable “BRCAness” phenotype. Thus, we sought to determine which combinations of standard CTx and PARPi might be synergistic promising therapeutic strategies for LMS. Methods: Dox, Docetaxel (Doc), Temozolomide (Tmz) were evaluated in combination with PARPi (Olaparib [Ola], Niraparib [Nira] and Talazoparib [Tala]) at 12 different drug concentrations. Four LMS cell lines of different origins (gynecological - GY, abdominal - A, extremity - E) were tested in a high throughput manner. All drug concentrations were chosen according to EC50. Cells were incubated with each combination for 7 days. Viability was assessed by ATPlite Luminescence Assay System.Evaluation of drug combination effect was performed using a Bliss synergy score. This system quantifies the degree of synergy as multiplicative effect of single drugs as if they acted independently. With a synergy score of -5 to 5, the interaction between two drugs is considered as additive; &lt;-5 antagonistic and &gt; 5 synergistic, and therefore a promising combination. Results: Anticancer activity, ranging from additive to synergistic was seen with all combinations. Results were consistent among all cell lines, independent of site of cell line origin (Table) Most synergistic combination in the majority of LMS cell lines were Dox or Tmz when combined with Tala, reaching up to 15 % and 27% above Bliss respectively. In contrast, Doc showed only additive effect with all analyzed PARPi. Conclusions: The data suggest that the combination of Dox or Tmz with PARPi may represent promising treatment options for LMS patients. Recent clinical studies support this notion in uterine LMS. Importantly these results suggest that such approach may be extended to all sites of LMS. Pre-clinical studies are underway to identify the most promising combinations for future clinical trial design. [Table: see text]

Pharmacologic Induction of BRCAness in BRCA-Proficient Cancers: Expanding PARP Inhibitor Use.

Simple SummaryBRCA1 and−2 are critical components of the homologous recombination pathway of DNA repair required to effectively repair DNA double strand breaks, leading to an increased cancer risk in patients with inherited BRCA mutations. An additional subset of cancers exhibit ‘BRCAness’, harboring repair defects stemming from mutations in non-BRCA DNA repair genes. Both BRCA-mutant cancers and cancers with a BRCAness phenotype are sensitive to PARP inhibitors, a class of cancer therapy drugs that inhibit the repair of DNA single strand breaks. To expand the use of PARP inhibitors to a larger group of patients, studies have focused on new combination strategies using agents that can induce BRCAness. This review focuses on the current status of drug-induced BRCAness in combination with PARP inhibitors to enhance cancer treatment.The poly(ADP-ribose) polymerase (PARP) family of proteins has been implicated in numerous cellular processes, including DNA repair, translation, transcription, telomere maintenance, and chromatin remodeling. Best characterized is PARP1, which plays a central role in the repair of single strand DNA damage, thus prompting the development of small molecule PARP inhibitors (PARPi) with the intent of potentiating the genotoxic effects of DNA damaging agents such as chemo- and radiotherapy. However, preclinical studies rapidly uncovered tumor-specific cytotoxicity of PARPi in a subset of cancers carrying mutations in the BReast CAncer 1 and 2 genes (BRCA1/2), which are defective in the homologous recombination (HR) DNA repair pathway, and several PARPi are now FDA-approved for single agent treatment in BRCA-mutated tumors. This phenomenon, termed synthetic lethality, has now been demonstrated in tumors harboring a number of repair gene mutations that produce a BRCA-like impairment of HR (also known as a ‘BRCAness’ phenotype). However, BRCA mutations or BRCAness is present in only a small subset of cancers, limiting PARPi therapeutic utility. Fortunately, it is now increasingly recognized that many small molecule agents, targeting a variety of molecular pathways, can induce therapeutic BRCAness as a downstream effect of activity. This review will discuss the potential for targeting a broad range of molecular pathways to therapeutically induce BRCAness and PARPi synthetic lethality.

The Next Frontier in Pancreatic Cancer: Targeting the Tumor Immune Milieu and Molecular Pathways.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) has a notoriously bad prognosis due to its high mortality and lack of good therapies. Chemotherapy is the current standard of treatment for PDAC, yet survival for most PDAC remain at around one year. Better therapeutic options are in dire need. Unlike other cancer types where targeted therapies and immunotherapies have changed the treatment landscape, their uses in pancreatic cancer are limited. However, there is increasing evidence in preclinical and early clinical studies that suggest these agents hold the key to the next frontier in PDAC treatment. We herein review some selected evidence.Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with abysmal prognosis. It is currently the third most common cause of cancer-related mortality, despite being the 11th most common cancer. Chemotherapy is standard of care in all stages of pancreatic cancer, yet survival, particularly in the advanced stages, often remains under one year. We are turning to immunotherapies and targeted therapies in PDAC in order to directly attack the core features that make PDAC notoriously resistant to chemotherapy. While the initial studies of these agents in PDAC have generally been disappointing, we find optimism in recent preclinical and early clinical research. We find that despite the immunosuppressive effects of the PDAC tumor microenvironment, new strategies, such as combining immune checkpoint inhibitors with vaccine therapy or chemokine receptor antagonists, help elicit strong immune responses. We also expand on principles of DNA homologous recombination repair and highlight opportunities to use agents, such as PARP inhibitors, that exploit deficiencies in DNA repair pathways. Lastly, we describe advances in direct targeting of driver mutations and metabolic pathways and highlight some technological achievements such as novel KRAS inhibitors.

RAD51AP1 and RAD54L Can Underpin Two Distinct RAD51-Dependent Routes of DNA Damage Repair via Homologous Recombination.

Homologous recombination DNA repair (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory HR sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54L, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that concomitant deletion of RAD51AP1 and RAD54L further sensitizes human cancer cell lines to treatment with olaparib, a Poly (adenosine 5′-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which induces DNA replication stress. We also show that the RAD54L paralog RAD54B compensates for RAD54L deficiency, although, surprisingly, less extensively than RAD51AP1. These results, for the first time, delineate RAD51AP1- and RAD54L-dependent sub-pathways and will guide the development of inhibitors that target HR stimulators of strand invasion.

PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development.

Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here, we show that protein phosphatase 4 catalytic subunit (PPP4C) deletion causes severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibited DNA lesions that inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of the essential HR factor RAD51. On the other hand, when inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods, which couple the checkpoint to HR.